Although sickle cell anemia is a genetic disease due to a hemoglobin mutation, a number of unique changes in the membrane of the sickle red blood cell have been identified. One of these is an alteration in phospholipid organization. Following sickling, phosphatidyl serine (PS), normally confined to the inner leaflet of the lipid bilayer, is present in the outer leaflet, exposed to the plasma environment. We hypothesize that this distortion of phospholipid organization, an event similar to that which occurs in the platelet membrane following activation, confers pro- coagulant properties on the red cell imbuing it with certain platelet-like characteristics in hemostasis. Based upon the membrane mass resulting from circulating red cells and vesicles released from red cells, our calculations suggest that the quantity of membrane PS available for hemostatic reactions either exceeds, or is equivalent to, that provided by activated platelets. Therefore, we hypothesize that the PS containing surfaces on red cells and vesicles in sickle cell disease can disrupt the hemostatic balance, result in a hypercoagulable state, and lead to specific vaso-occlusive complications. Our goal in this proposal is to investigate the mechanisms responsible for, and hemostatic consequences resulting from, loss of phospholipid asymmetry in subpopulations of sickle cells and red cell derived vesicles. Furthermore, we will investigate the hypothesis that red cell derived membrane surfaces containing PS activate clotting and contribute to the pathophysiology of sickle cell disease. To pursue these goals, we have developed three specific aims: l. To investigate the mechanisms responsible for loss of phospholipid (PS) asymmetry in sickle red blood cells, 2. To determine if sickle red cells and vesicles generated from sickle red cells cause a "hypercoagulable" state in sickle cell disease, 3. To correlate the amount of PS- erythrocyte-derived-bilayer-surface in the peripheral circulation with measurements of hypercoagulability and clinical manifestations of sickle cell disease. A new method using fluorescent annexin V and flowcytometry will be used to identify and isolate individual cells with an abnormal phospholipid organization. PS exposure will be confirmed with an in vitro prothrombinase assay. Cells with an abnormal phospholipid organization will be characterized by (confocal) fluorescent microscopy and selected from the population by magnetic bead separation. Transbilayer movement of phospholipids will be determined by the movement of spin labeled phospholipids. Hemostatic footprints of PS exposure in vivo will be determined by standard assays for prothrombin fragment 1+2 (F1.2), thrombin anti thrombin complex (TAT) and Fibrinopeptide A (FPA). Results will be correlated with the clinical manifestations of sickle cell disease in particular in patients with acute chest syndrome (ACS) and patients identified by Transcranial Doppler (TCD) to be at high risk for developing subsequent stroke Our research findings should provide new information on the mechanism of loss of phospholipid asymmetry sickle cells, and its potential physiologic effects.